This invention relates to a process for resolving mixtures of D,L-leucine. Specifically, N-acyl-D,L-leucine ester mixtures can be selectively enzymatically hydrolyzed and, as a result, separated into the D- and L-optical antipodes.
Synthetic, racemic D,L-amino acids can be utilized under conditions which vary with the case in question. Man can utilize the D-forms of amino acids as a racemic mixture and by conversion of D-form to L-form. However, various animals have varying utilization efficiencies for the D- or L-forms of amino acids. Specifically, the rat and the mouse utilize L-leucine alone, whereas, in the chick, D,L-leucine and L-leucine are equally well utilized for growth.
It should also be noted that for growth, maintenance, reproduction and tissue repair, organisms require a supply of qualitatively and quantitatively specific amino acids. One of the essential amino acids is leucine. As a racemic mixture, it can be used by the body, but in its L-form it is more useful to combat malnutrition and for supplementation of a deficient diet. Thus, there is a need for processes which conveniently resolve racemic mixtures of D,L-leucine.
Several principles have been applied in the resolution of D,L-forms of amino acids into their D and L components. Conventional procedures for such separations include (1) direct crystallization, for example, by seeding a highly concentrated solution of D,L-threonine with a known amount of L-threonine, the L-form will crystallize out; (2) crystallization with an optically active reagent in polarized light, for example, the alkaloids, bases and optically active reagents in polarized light can form derivatives having different solubilities; and (3) enzymatic methods. In this last method, some enzymes behave differently with respect to the D and L-forms of amino acids. Thus, orginase acts only on the L-isomer, the D-isomer remaining intact in the solution from which it may then be extracted. Also, the L-amino acid oxidases may be used to extract D-leucine, D-methionine and D-phenylalanine. Kirk-Othmer, Encyclopedia of Chemical Technology, Second Edition, Vol. 2, John Wiley & Sons, Inc., New York, pp. 156-197.
Specifically, a number of methods for the optical resolution of D,L-leucine are known. For example, the physicochemical resolution by preferential crystallization of either one of the optically active isomers from a supersaturated solution of acetyl-D,L-leucine is known. In the case of enzymatic resolution, acetyl-D,L-leucine can be asymmetrically hydrolyzed by treatment with aminoacylase obtained from hog kidney or moulds. Optical resolution is then possible in good yield by separating the resultant products, L-leucine and acetyl-D-leucine. Such asymmetric hydrolysis has also been achieved with insolubilized mould aminoacylase. Kaneko et al, Synthetic Production and Utilization of Amino Acids, J. Wiley & Sons, New York (1974), p. 136-140.
In U.S. Pat. No. 3,963,573, there is taught a process for producing optically pure N-acyl-L-methionine by subjecting an N-acyl-D,L-methionine ester to the action of a proteolytic enzyme selected from the group consisting of sulfhydryl proteinases and microbially derived serine proteinases and separating the resulting N-acyl-L-methionine. The art has recognized that certain proteolytic enzymes can be produced in a pure form, such as from Bacillus subtilis, Guntelberg Trav. Lab. Carlsberg, Ser. Chim. Vol. 29, p. 36-48 (1954). The proteolytic enzyme prepared from the strain of Bacillus subtilis was purified by crystallization and its physico-chemical properties were determined. The enzymatic properties were investigated insofar as optimum pH for milk coagulation, stability, degradation of casein, hydrolysis of hemoglobin, activators and inhibitors for the enzymes, the effect on ovalbumin and other characteristics. In the Journal of Biological Chemistry, Vol. 243, No. 7, pp. 1344-1348 (1968) Barel, examined the activity of Carlsberg and Novo subtilisins toward a number of N-acetylamino acid esters and amino acid esters. The enzymes were also compared with respect to their efficiency in catalyzing aminolysis reactions, their rates of inactivation by certain aromatic sulfonyl halides and the rates of deacylation of their N-trans-cinnamoyl derivatives. Although the enzymes were found qualitatively indistinguishable from the standpoint of substrate specificity, significant quantitative differences were observed. Thus, the microbially derived serine proteinases, for example, Novo and Carlsberg subtilisin exhibited varying of degrees of esterase activity on various N-acyl-L-amino acid esters. However, there is not disclosed a process for resolution of N-acyl-D,L-leucine esters employing the activity of serine proteinases. An especially effective process would involve (1) readily available and inexpensive material, such as enzyme, and (2) produce high purity material in high yield at a rapid rate.